Endothelial MicroRNA-214 Confers Angiotensin II Hypertension by Targeting eNOS in Mice Open Access

Hypertension is a complex cardiovascular disorder and affects approximately 31% of the world adult population [1]. Epidemiological studies have shown that clinical morbidity and mortality associated with hypertension are emerging as a major health challenge of the 21st century [2]. Hypertension is closely associated with increased cardiac output or peripheral vascular resistance. At the same time, hypertension appears as an independent risk factor of cardiovascular disease, often accompanied by endothelial dysfunction caused by abnormal endothelial cell (EC) activation, which may be a process or result of hypertension [3‒8]. EC dysfunction is involved in hypertension pathogenesis in a variety of ways, including vasodilator dysfunction [9‒12], reduced nitric oxide bioavailability [10, 12, 13], inflammation [14, 15] and secretion of coagulants [16], and deficiency in angiogenesis, leading to scarce capillaries [17]. A downregulation of vascular endothelial NO synthase (eNOS) has been shown in multiple hypertension animal models [18, 19]. Hypertension in eNOS-null mice provided solid evidence, demonstrating that eNOS deficiency contributes to the development of high blood pressure [10, 20]. However, despite the compelling evidence for the role of eNOS activity in modulating blood pressure, mechanistic studies on the regulation of eNOS and hypertension are still required.

MicroRNAs are important small noncoding RNAs that negatively regulate gene expressions in the translational step. Studies reported that the changes of several miRNAs in blood and the dysregulation of some miRNAs in tissues are linked to CVDs [21‒25]. Further evidences indicated that the dysregulation of miRNAs could serve as causative factors in the pathogenesis of endothelial dysfunction by influencing eNOS expression, endothelial repair, angiogenesis of blood vessel walls, or secretion of inflammatory molecules [11, 26‒28]. In mouse cardiac tissue, many miRNAs were found to be up- or downregulated in response to transverse aortic constriction [29‒31]. Among them, miR-214 was considered as a marker of the stress [31‒33]. Recent reports have also shown that miR-214 is related to the occurrence of cardiac fibrosis [34‒38], myocardial damage [39‒42], angiogenesis [43‒47], and inflammatory reaction [48‒51]. More interestingly, some studies revealed that the abnormal expression of miR-214 is also involved in the occurrence and development of pulmonary hypertension [52‒54]. However, whether miR-214 plays an important role in the occurrence and development of Ang II-induced hypertension remains unclear.

To verify the effect of miR-214 in the occurrence and development of Ang II hypertension, we broadly validated the function of miR-214 in blood pressure regulation employing vascular EC-, smooth muscle cell-, and renal tubular cell-miR-214-specific knockout mice, and ECs challenged with Ang II, agomiR-214, and antagomiR-214. These findings not only confirm that miR-214 might play an important role in blood pressure regulation by directly targeting eNOS but also provide a new target for the prevention and treatment of hypertension.

Fetal bovine serum, penicillin-streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), and trypsin solution (EDTA) were bought from Gibco (Invitrogen, Grand Island, NY, USA). Ang II (Cat #: A9525) was bought from Sigma (St. Louis, MO, USA). Antibody against total eNOS and phospho-Ser1177 of eNOS were all from Abcam (Cat #: ab199956, ab215717). Mouse antagomir-214, miR-214 inhibitor, agomir-214, and miR-214 mimics were purchased from GenePharma Co., Ltd. (Shanghai, China).

miR-214^fl/fl mice with 129 genetic background were constructed by the Model Animal Research Center of Nanjing University (Nanjing, China). Tek-Cre (also known as Tie2-Cre, stock no. 004128), Tagln-Cre (also called Sm22a-Cre, stock no. 017491), and Kap-Cre (stock no. 008781) transgenic mice were purchased from JAX^® Mice (Bar Harbor, ME, USA). To generate mice with miR-214 deletion in vascular endothelial (Tek-Cre), smooth muscle (Tagln-Cre), and renal proximal tubule cells, mice carrying floxed miR-214 were bred with Tek-Cre, Tagln-Cre, and Kap-Cre mice. After backcrossing, miR-214^fl/fl/Tek-Cre, miR-214^fl/fl/Tagln-Cre and miR-214^fl/fl/Kap-Cre mice were obtained. Mice with miR-214 deletion in ECs, smooth muscle cells, and renal proximal tubule cells were of normal size and body weight and did not display any apparent vascular and renal pathology under normal condition.

All the mice were kept in a heated room and subjected to a 12-h circadian cycle. Genotyping was performed using tail DNA. Endothelial cell, smooth muscle cell, and renal proximal tubule cell miR-214 cKO mice and their littermate wild-type (WT) control mice aged 2–3 months (male) were used in experiments.

For miR-214 antagomir and agomir studies, 8-week-old C57/BL6 male mice obtained from the Animal Research Center of Nanjing Medical University were randomly assigned into groups as follows: The miR-214 antagomir and agomir provided by GenePharma (Shanghai, China) were administered intraperitoneally at a dose of 10 mg/kg in 200 μL saline, respectively. All procedures were carried out in accordance with the standard SOP formulated by the Experimental Animal Ethics Committee of Nanjing Medical University.

Mice were placed in an individual metabolic cages with stainless steel grid floor, 23 cm in diameter and 18 cm high (specially designed cage with a collection channel at the bottom). Twenty four-hour urine from the collection channel was collected into a sterile EP tube. The mice were supplied with water and standard pelleted diet [55].

Urinary sodium (Na+), potassium (K+), and chloride (Cl−) were analyzed by ion-selective electrode on automatic electrolyte analyzer (EasyLytePLUS Na+/K+/Cl−, Medica Corporation, USA). Four hundred microliters of diluted (1:10) urine was placed into the sample detector, and the analyzer automatically performs the analysis and records the results [56].

Aorta vessels (approximately 20 cm in length) from anesthetized mice were placed in precooled DMEM. The vascular fat and the connective tissue were cleaned under a microscope. After opening the vessels longitudinally, the ECs were gently scraped on the surface of the intima with a scraper. Then, the extravascular membrane was removed, and the layer of vascular smooth muscle cells was kept for experimental analysis [57].

Under isoflurane anesthesia (1.5%), mice were placed through a median incision in the scapula, and a micro-osmotic pump (ALZET, model 1002) filled with Ang II was implanted subcutaneously. This micro-osmotic pump can release a dose of 1.4 mg/kg of Ang II dissolution at a rate of 0.25 μL/h per day.

Firstly, the systolic blood pressure (SBP) in the tail vein of mice was tested by the tail-cuff method using the Visitech BP 2000 Blood Pressure Analysis System (Apex, NC, USA) [58]. All the animals were accustomed to using the blood pressure measuring device for 7 days, and then each animal was measured 15 times in 2 cycles per day. Then, a telemetric device was employed to detect the mean arterial pressure (MAP) [59, 60]. According to the instructions of the physiological telemetry device (model no. TA11PA-C20; DSI, St Paul, MN, USA), the catheter was implanted into the carotid artery of the mice. Then, the telemetry probe was turned on about 1 week after the mice recovered and the basic MAP was measured before Ang II perfusion.

The mouse aorta endothelial cells (MAECs) were purchased from Jennio Biotech Co., Ltd. (Guangzhou, China). The cells were cultured in DMEM with 10% fetal bovine serum and 5% CO₂ at 37°C. After MAECs were cultured to 60%–70% density, the Lipofectamine 2000 Kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect miR-214 inhibitors (80 nm), mimics (40 nm), and their controls for 48 h, and then the cells were collected for subsequent use.

Total RNA was isolated from aortic tissues or MAECs using TRIzol reagent according to the instructions (Invitrogen; Thermo Fisher Scientific, Inc.). Relative miR-214 expression was detected on the Applied Biosystems Q3 quantitative PCR instrument (Thermo Fisher Scientific, Inc.) by using an SYBR PrimeScript miRNA RT-PCR kit (Takara Bio, Inc., Otsu, Japan), and U6 was normalized for the aorta and cell samples. The Bulge-Loop™ miRNA qRT-PCR primer sets for miR-214 were synthesized by RiboBio (Guangzhou, China). eNOS expression was detected by qRT-PCR using the Applied Biosystems Q3 system. GAPDH was used as a reference gene. The primer sequences of eNOS and GAPDH were as follows: eNOS forward, 5′-CGT​CCT​GCA​AAC​CGT​GCA​GA-3′ and reverse, 5′-TCC​TGG​GTG​CGC​AAT​GTG​AG-3′; and GAPDH forward, 5′-GTC​TTC​ACT​ACC​ATG​GAG​AAG​G-3′ and reverse, 5′-TCA​TGG​ATG​ACC​TTG​GCC​AG-3′. The PCR procedure consists of predenaturation (95°C, 10 min) followed by 40 cycles (95°C, 15 s, 60°C, 1 min). The data were analyzed by using the 2−ΔΔCq method [61].

TargetScan (http://www.targetscan.org/index.html) and miRanda (http://www.microrna.org) can be used to predict the target genes of miR-214. WT (PmirGLO-NOS3 3′-UTR-WT) and mutant (PmirGLO-NOS3-3′-UTR-Mut) plasmids were constructed by GenePharma Co., Ltd. (Shanghai, China). MAECs were transfected with PmirGLO-NOS3-3′-UTR-WT or PmirGLO-NOS3-3′-UTR-Mut with miR-214 mimics or negative control. Renilla luciferase plasmid (pGL4.73, Promega) was used as control for luciferase activity. After 48 h transfection, luciferase activity was measured by the luciferase reporter gene assay system (Promega Corporation).

After transfection for a certain time, the MAECs or mouse aortas were lysed with lysis buffer containing protease inhibitor. The concentration of related proteins was detected by using Micro BCA protein assay kit (Pierce, Thermo). The proteins were isolated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad). The membrane was enclosed in TBST containing 5% buttermilk at room temperature for 1 h and then incubated with eNOS (1:1,000) and phospho-Ser1177 (1:1,000) primary antibody at 4°C overnight, and with HRP-labeled secondary antibodies for 1 h at room temperature. Then, the images were examined with imaging equipment (The ChemiDoc XRS+ System, Bio-Rad), and the band intensity was measured with Image J software (NIH, Bethesda, MD, USA).

Urine samples were centrifuged at 10,000 rpm for 5 min, and then the urine supernatant was diluted to 1:50 with enzyme immunoassay buffer. Urine albumin was determined using an ELISA Starter Accessory Kit (Bethyl).

Urine NO was detected by using a Nitric Oxide assay kit (S0023, Beyotime). Urine samples were diluted to 1:10 and added to 5 μL NADPH (2 mm), 10 μL of FAD, and 5 μL of nitrate reductase for 30 min at 37°C. Then, 10 μL of LDH buffer was added in the above mixture for another 30 min at 37°C. Finally, the mixture was mixed with 50 μL of Griess reagent I and II for 10 min at the room temperature. Multimode microplate reader (Synergy^™ H1, Biotek) was performed to measure the absorbance of each well at 540 nm [62].

Data were expressed as mean ± SEM. Statistical differences among groups were analyzed by one-way ANOVA and multiple comparisons, and the significant difference between groups was analyzed by Tukey's post hoc test. Two-way ANOVA and mixed models were used for the comparison of the time-series data. A two-tailed unpaired Student’s t test was used to determine differences between two groups. All the statistics and data analysis were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). p < 0.05 was statistically significant.

To evaluate the potential relationship between the miR-214 and hypertension, we detected the effects of Ang II on miR-214 expression in mice and MAECs. The results showed that Ang II significantly increased the expression of miR-214 in aortic cells and ECs, suggesting that miR-214 may play an important role in the regulation of blood pressure (Fig. 1a, b). To verify this hypothesis, miR-214 antagomir was administered to Ang II hypertension mice. Three days of Ang II infusion markedly elevated the blood pressure of mice in both groups detected by the tail-cuff method (Fig. 1c). Then, mice were treated with miR-214 mimics by intraperitoneal injection. Strikingly, the level of blood pressure was significantly reduced by around 20 mm Hg in mice with miR-214 antagonism (Fig. 1c). Meanwhile, the expressions of eNOS and p-eNOS were elevated in miR-214 antagomir-treated mice compared to anti-control-treated animals (Fig. 1d, e). These data indicated that miR-214 could regulate blood pressure possibly through regulating eNOS to some extent.

It is known that eNOS in ECs is important in regulating blood pressure. In consideration of a potential effect of miR-214 on direct regulation of eNOS in ECs, we generated EC-specific miR-214 cKO mice using a Cre-loxP system to examine the role of miR-214 in blood pressure regulation. The genotypes of all offspring were analyzed by PCR (Fig. 2a). For all subsequent experiments, we used two genotypes: miR-214^fl/fl/Tek-Cre (−) herein designated as control and miR-214^fl/fl/Tek-Cre (+) herein designated as the vascular EC miR-214 null. As shown in Figure 2b and c, miR-214 was selectively deleted in ECs by approximately 70% without affecting miR-214 in smooth muscle cells of the artery as compared with littermate control mice. At the same time, compared with the control ones, endothelium-specific knockout of miR-214 did not affect the body weight, aortic morphology, and basal SBP of mice (Fig. 2d–f).

We then detected the response of endothelial miR-214 cKO mice to Ang II treatment. As shown in Figure 3a, SBP was significantly elevated in control mice during Ang II permeation detected by the tail-cuff method, while endothelial miR-214 deletion remarkably attenuated such a hypertensive response. Furthermore, daily MAP was measured using a telemetry system. Before Ang II treatment, we found that the basal MAP was similar between genotypes (Fig. 3b). After monitoring basic MAP, the mice were administered with Ang II for 7 days. Continuous infusion of Ang II significantly increased MAP in mice. Moreover, the increment of MAP in endothelial miR-214 knockout mice was markedly blunted by around 20 mm Hg compared with the littermate controls (Fig. 3b). Similar results were obtained in the SBP and diastolic blood pressure (Fig. 3c, d). To further reveal the nature of blood pressure phenotypes in these animals, we detected protein levels of eNOS and phosphorylated eNOS. Strikingly, both eNOS and p-eNOS levels were markedly higher than that in the WT mice after Ang II infusion (Fig. 3e, f). Then, we examined the NO in urine and found that endothelial-specific miR-214 knockout mice had higher levels of NO in the urine (Fig. 3g). Furthermore, we examined proteinuria associated with Ang II hypertension and miR-214 inhibition. As shown in Figure 3h, proteinuria was attenuated in endothelial miR-214 cKO mice as compared with the control ones after Ang II treatment, which suggested a renal protective effect of miR-214 antagonism against hypertension-induced kidney injury. These results indicated that the endothelial miR-214 play a crucial role in Ang II-induced hypertension by targeting eNOS.

To better evaluate the miR-214 effect on regulating eNOS in vivo, we intraperitoneally injected miR-214 agomir or miR-214 antagomir into the mice. As expected, miR-214 expression was markedly elevated in the aortas of mice received miR-214 agomir treatment (Fig. 4a). Meanwhile, the expressions of eNOS and p-eNOS were decreased as compared with the mice treated with negative control (Fig. 4b–d). Then, the SBP was significantly elevated about 20 mm Hg in mice with miR-214 agomir (Fig. 4e). In contrast, injection of miR-214 antagomir reduced vascular miR-214 but elevated the expressions of eNOS and p-eNOS (Fig. 4f–i). As expected, the SBP was significantly suppressed after treatment with miR-214 inhibitors (Fig. 4j). In vitro, we transfected the miR-214 mimics into the MAECs and found that overexpressed miR-214 significantly decreased the expression level of eNOS and p-eNOS (Fig. 5a–d). In contrast, knockdown of miR-214 using miR-214 antagomir increased the expressions of eNOS and p-eNOS (Fig. 5e–h). Then we analyzed the possible targets of miR-214 by TargetScan and miRanda search tools and found that eNOS may be the possible target gene of miR-214 as shown by a complementarity between eNOS 3′-UTR and miR-214 (Fig. 5i). Finally, eNOS was found to be the target gene of miR-214 by luciferase reporter gene detection (Fig. 5j). These results confirmed the direct regulation of eNOS by miR-214 in vivo and in vitro.

The main physiological roles of arterial smooth muscle cells include growth, contraction, and relaxation. In consideration of the important role of vascular smooth muscle cells in blood pressure regulation, the Cre-loxP system was also used to conditionally knock out miR-214 in VSMCs. The schematic representation of the floxed miR-214 allele and the breeding strategy used was similar as endothelial specific miR-214 cKO mice (Fig. 6a). miR-214 was selectively deleted in the smooth muscle of the artery by approximately 90% without affecting miR-214 in endothelium as compared with littermate control ones (Fig. 6b, c). Meanwhile, there was no significant difference in body weight and aorta morphology between genotypes (Fig. 6d, e). Furthermore, a tail-cuff measurement was applied to detect the SBP before and after Ang II treatment. As shown in Figure 6f, the SBP was similarly increased after Ang II treatment between genotypes, indicating that miR-214 in vascular smooth muscle might play no role in the occurrence of Ang II hypertension.

We further investigated whether specific knockdown of miR-214 in proximal renal tubule epithelial cells mitigated Ang II-induced hypertension. Renal proximal tubule miR-214 conditional knockout mice (miR-214^fl/fl/Kap-Cre) were generated according to the strategy as our previous study [63]. The SBP between WT and renal proximal tubule-specific miR-214 cKO mice was monitored by the tail-cuff method. As shown in Figure 7a, there was no difference in SBP after Ang II infusion between genotypes. Furthermore, metabolic studies were used to compare electrolyte balance in control and renal proximal tubule-specific miR-214 cKO mice with Ang II-induced hypertension. Metabolic cages were used to collect 24-h urine samples of mice before and after Ang II treatment. Urine volume (Fig. 7b), urine output of sodium (Fig. 7c), potassium (Fig. 7d), and chloride (Fig. 7e) were all elevated at the similar levels between the two groups after Ang II treatment. Meanwhile, there was no significant difference in body weight between genotypes (Fig. 7f). These results suggested that miR-214 in real proximal tubules might play no role in the occurrence of Ang II hypertension.

Hypertension is related to endothelial dysfunction and increases morbidity and mortality. It is well known that blood pressure regulation is a rather complex and unclear process. In general, the central nerve system, kidney, cardiovascular system, and metabolic and local nervous factors are all associated with the regulation of blood pressure. However, the causes and detailed mechanisms of hypertension are still uncertain. Very interestingly, recent studies indicated that miR-214 plays a critical role in hypoxic pulmonary hypertension [52‒54, 64]. Most recently, a study indicated a prohypertensive function of renal miR-214-3p in salt-sensitive hypertension [65]. In addition, Nosalski et al. [66] found that serum-circulating miR-214 levels were higher in hypertensive patients and miRNA-214 from T cell-mediated hypertensive perivascular fibrosis. However, the vascular miR-214 in hypertension still needs further investigation. Here, we demonstrated an increase of miR-214 level in Ang II-induced aortas of mice, which suggests that miR-214 has a potential role in controlling blood pressure. To test whether miR-214 could affect blood pressure, the miR-214 antagomir was administered to the mice via IP injection after Ang II infusion. Strikingly, anti-miR-214 significantly attenuated Ang II hypertension. Importantly, the miR-214 effect on blood pressure was accompanied by a regulation of eNOS and p-eNOS in vasculature.

It is known that endothelial NOS is predominantly expressed in the endothelium and controls the vascular tone via the synthesis of nitric oxide. To evaluate the function of endothelial miR-214 in the regulation of blood pressure and eNOS, we generated EC-specific miR-214 cKO mice. This cKO mouse line did not show the obvious defects in baseline phenotypes, including body growth, vascular morphology, and blood pressure. Furthermore, we established an Ang II hypertension model using WT and cKO animals and observed a significant attenuation of hypertension in endothelial miR-214 cKO mice verified by telemetry and tail-cuff methods. Along with the improved hypertension in endothelial miR-214 cKO mice, the proteinuria was also attenuated, suggesting an amelioration of Ang II hypertension-induced kidney injury. These data demonstrated that endothelial miR-214 also potently contributed to Ang II hypertension.

Furthermore, we observed that inhibition or activation of miR-214 in animals and ECs distinctly regulated the expressions of eNOS. Based on above data, we speculated that eNOS might be one of the target genes of miR-214. Consistent with this hypothesis, bioinformatic analysis showed that miR-214 could regulate eNOS transcription by directly targeting eNOS mRNA. By a luciferase assay, we confirmed that eNOS is the target gene of miR-214. Those results highly indicated that miR-214 might regulate the systemic blood pressure probably via a negative modulation of eNOS in vasculature to some extent.

By now, many genes have been confirmed as direct targets of miR-214 in many disease models including cardiovascular diseases. For examples, PTEN in right ventricular [31] and cardiomyocyte survival [47], ing4 and EZH2 in cardiomyocytes [67], QKI in vascular angiogenesis [46], ITCH in viral myocarditis [68], and COX-2 in EC apoptosis [69] were documented as the target genes of miR-214. These data suggested that miR-214 can be regulated in response to different stimuli and acted in a tissue-specific manner possibly because of the specificities of tissues and stimuli. In ECs, our data indicated that miR-214 could target eNOS to modulate blood pressure. However, beyond eNOS, the downstream targets of miR-214 also involved ATF4 [70] and PI3K/AKT/mTOR axis [71], etc., contributing to oxidative stress and inflammation. Both oxidative stress and inflammation also play roles in hypertension, which needs further investigation in this experimental setting in the future.

Vascular smooth muscle cells play a crucial role in maintaining vascular tension and integrity. VSMCs exhibit significant phenotypic regulation under disease conditions. The proliferation and migration of VSMCs contribute to the pathological process of vascular disorders, including hypertension, restenosis, and atherosclerosis [72‒76]. Accumulating evidence implicates that microRNAs in VSMCs could modulate the differentiation, phenotypic switching, and the maintenance of the contractile phenotype. In particular, loss of the miR-143/145 cluster in VSMCs resulted in deregulated blood pressure in normolipidemic mice [77‒79]. In vivo studies demonstrated that inhibiting miR-30c-5p in smooth muscle cells blocks cocaine-induced increment of blood pressure [80]. Hence, we generated another mouse line with a specific knockout of miR-214 in VSMCs. Mice with miR-214-specific knockout in VSMCs exhibited normal growth, vascular morphology, and basal blood pressure. Following angiotensin II (Ang II) infusion, both WT- and VSMC-specific miR-214 KO mice generated paralleled hypertension, suggesting a specific role for endothelial miR-214 in promoting Ang II-induced hypertension. However, a recent study by Li et al.’s [81] group reported that miR-214 in VSMCs promoted Ang II-induced hypertension [81], which disagreed with our findings. After reviewing their publication, we found that the discrepancy between the two studies could be due to the genetic differences in the animals (C57BL/6J background in Li’s study vs. 129 background in our study). Moreover, the detailed strategy of miR-214 deletion in VSMCs might not be the same, which needs further validation with Li et al.’s [81] group. Besides, Nosalski et al. [66] generated systemic KO of miR-214 in mice and they did not find effect of Ang II on BP within the first 7 days. Such a discrepancy could be related to the multiple cellular and molecular targets of miR-214. Systemic deletion of miR-214 could affect the development or function of other types of cell and organs, which might lead to phenotype difference under Ang II challenge. However, the details need further investigation. Our group also generated the renal proximal tubule-specific miR-214 cKO mice and found a similar increment in blood pressure after Ang II infusion. Consistently, the urinary output of electrolytes showed no difference between genotypes before and after Ang II infusion, suggesting that miR-214 in the proximal tubules played no role in regulating Ang II hypertension. This discrepancy between studies may be due to differences in hypertension models, the nonrenal effects of anti-miR-214 delivery via renal indwelling catheter, and the effects of distal nephron miR-214. In the future, the function of miR-214 in regulating blood pressure in other nephron segments needs to be further determined.

In summary, employing animals treated with miR-214 agomir/antagomir, three strains of conditional miR-214 knockout mice challenged with Ang II, as well as the in vitro ECs, we reported that miR-214 in ECs but not in smooth muscle cells and renal tubular cells was a potent regulator of blood pressure possibly by targeting eNOS. The results of this study verified a critical role of EC miR-214 in regulating blood pressure, which increased our understanding on the pathogenesis of hypertension.

This study was approved by the Laboratory Animal Ethics Association of Nanjing Medical University (No. 2102005-1) and all animal experiments are conducted in accordance with regulations on the administration of experimental animals and relevant national laws and regulations.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by Grants from the National Natural Science Foundation of China (Nos. 82070702 and 81770740), the National Key Research and Development Program (No. 2019YFA0802702-1), Social Development Fund of Jiangsu Province (No. BE2021607), the “333” talent plan of Jiangsu province (No. 333-2022001), and Nanjing City Key Medical Research Project (No. ZKX24036).

Shuzhen Li: methodology, investigation, and writing – original draft and review and editing. Bing Liu: validation and data curation. Shuang Kang and Bingyu Yang: validation and methodology. Yue Zhang and Songming Huang: validation and investigation. Aihua Zhang and Zhanjun Jia: conceptualization, funding acquisition, and writing – review and editing.

Shuzhen Li and Bing Liu contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.